Bacterial chemotaxis is a virulence factor for pathogenic bacteria and is a model for recognition of, and response to chemicals in unicellular and multicellular organisms. The proposed research extends work done under the previous grant and expands it into new areas. Our study of the interaction between periplasmic maltose-binding protein (MBP) and the Tar chemoreceptor will focus on structural studies and modeling. The question of whether covalent adaptation, like transmembrane signaling, is asymmetric between the two subunits of the Tar homodimer will be addressed, as will the structural basis for the inability of Tar from Salmonella to mediate maltose taxis. Hypotheses about the role of tryptophan residues at the borders of transmembrane helix II of Tar will be tested with site- directed mutagenesis. We will also analyze interaction of the dipeptide-binding protein (DppA) and its cognate receptor, Tap, in genetic studies designed to define the mechanisms by which binding-protein/transmembrane-receptor pairs function in chemotaxis. We have created a chimera between the NarX sensor kinase and Tar that is a repellent receptor for nitrate/nitrite. We will generate the reciprocal Tar/NarX fusion to determine how its kinase activity is regulated by Tar attractants and repellents. We will also create reciprocal chimeras between Tar and other sensor kinases, such as the osmosensor EnvZ of E. coli and Salmonella and the cell-density sensor SasS of Myxococcus xanthus. These hybrids will provide further information on mechanisms of transmembrane signaling, allow analysis of the ligand-recognition properties of the sensor kinases, and produce sensors that can be used to engineer bacteria to be detectors of environmental chemicals. Finally, we are interested in dynamic complexes that form between membrane receptors and soluble signaling proteins. Phosphorylation systems reconstituted in vitro will combine multiple receptors, CheW coupling factor, CheA kinase, and CheY response regulator to monitor receptor cross- talk. This issue will be addressed in vivo by comparing chemotaxis in cells in which different receptors are expressed sequentially versus simultaneously. The demonstration that fusions between CheZ and fluorescent proteins (GFP or YFP) retain CheZ function and subcellular localization suggests that these constructs can be used to probe the factors that affect localization of CheZ to the chemoreceptor patch, to assess the significance of CheZ localization in signal transduction, and to examine the sequence of events that leads to formation of the patch in growing and dividing wild-type cells and cell-division mutants. The supramolecular architecture of the receptor patch and its associated proteins will be examined using electron tomography.